It has been suggested that cell death not only plays an important role in basic control of the nervous system, the endocrine system and the immune system in higher organisms but also is deeply involved in many diseases (Thompson C. B., Science, vol. 267, p. 1456-1462 (1995). Some diseases including, for example, autoimmune diseases such as systemic lupus erythematosus, neurodegenerative diseases due to death of neurons, organ transplantation injuries associated with organ transplantation, etc. may be regarded as one due to influence of cell death where apoptosis is involved.
Factors causing cell death includes both an extraneous factor and an intrinsic factor. For an extraneous factor, those of which substantial existence as substance accelerating cell death have been established include TNF involved in the immune system (Zheng, L., et al., Nature, vol. 377, p. 348-351 (1995)), Fas ligand (Suda T., et al., Cell, vol. 75, p. 1169-1178 (1993)), glucocorticoids (Wyllie A. H., Nature, vol. 284, p. 555-556 (1980)), etc. An extraneous factor also includes lack of a growth factor indispensable to cell growth, such as erythropoietin, interleukins, nerve growth factor, or lack of nutritional factors. In these cases, cell death is induced by apoptosis caused by change in physiological conditions. Apoptosis may also be induced by non-physiological stresses such as radiation, temperature, anticancer agents, calcium ionophore, active oxygen, etc. In addition, necrosis may also be induced by burn, toxic substance, ischemia, attack by complements, infection with virulent virus, administration of overdose medicaments or overdose radiation.
For an intrinsic factor, there are changes in the metabolic system such as intracellular concentration of Ca2+, metabolism of nucleic acids, metabolism of amino acids, metabolism of energy, etc., which lead to cell death. Control of these apoptotic signals could have lead to protection from exacerbation of conditions of, prevention or treatment of various diseases. However, at present, the mechanism is not so simple that mere control of the causal substance and factors that have hitherto been established cannot afford sufficient clinical application.
On the other hand, as substance that have hitherto been proved to inhibit cell death, intracellular factors such as bcl-2 and bcl-x are known that are believed to inhibit most of apoptotic signals (Boise L. H., et al., Cell, vol. 74, p. 597-608 (1993)). However, these agents must intracellularly be expressed for causing inhibition of cell death and effects can hardly be obtained by extracellular addition of these agents. Extracellular factors for inhibiting cell death have also been reported that inhibit apoptosis by active oxygen, including superoxide dismutase (hereinafter also referred to as “SOD”) (Greenlund L. J., et al., Neuron, vol. 14, p. 303-315 (1995)), catalase (Sandstrom P. A. and Buttke T. M., Proc. Natl. Acad. Sci. USA, vol. 90, p. 4708-4712 (1993)), and glutathione peroxidase (Kayanoki Y., et al., J. Biochem., vol. 119, p. 817-822 (1996)). However, cell death cannot effectively be inhibited by these extracellular factors alone.
While culturing cells, cell death is induced due to stress to cells imposed by substances from the cultured cells per se or from extraneous additives. However, it is not all the cells that are put to death under certain conditions. For those cells that survived the circumstances, proteins necessary for suppressing the cell death-inducing signals due to stress under their thresholds should have already been expressed, or newly induced, either intracellularly or extracellularly. Such proteins include, as envisaged, transcription factor, synthases, enzymes related to metabolism, oxidases, reductases, kinases, transferases, apoptosis-inhibiting proteins, etc. That is, sensitivity to stress in each of respective cells may vary due to difference in their expression level of these proteins. Thus, even if the mechanisms of cell death are not always the same, if the cell death-inducing signals could be suppressed under their thresholds by extraneously adding an inhibitory agent to cell death due to certain stress, then cell death could possibly be inhibited not only in cultured cells but also within the living body where similar stress occurred.
Moreover, cell death is closely related to diseases. Thus, identification of a number of agents having a cell death-inhibitory activity within the living body to control a variety of cell deaths would not only allow for clinical application such as treatment of diseases but also for application to effective culture of cultured cells. Indeed, although some factors are known that inhibit cell death, e.g. bcl-2, bcl-x, etc. as intracellular cell death-inhibitory factors, or SOD, catalase, glutathione peroxidase, etc., as extracellular factors, it is difficult to inhibit cell death in all types of cells by extracellular addition of these factors. This is due to difference in processes through which cell death is mediated based on difference in their mechanisms. Taking this into consideration, there is a need to identify activities that significantly, and more specifically, inhibit a variety of cell deaths. That is, for those cell deaths that are not subject to inhibition by known materials, there is a need to searching for factors that can significantly inhibit said cell deaths. In addition, cell death-inhibitory factors are likely to be present for maintaining homeostasis within the living body and hence identification of such factors is extremely significant.
While culturing cells under cell-free conditions or another special conditions, apoptosis induced by stress is frequently observed. Cell culture is performed under these cell death-inducing conditions and with the index of the cell death-inhibitory activities effective components in blood may be purified by using various chromatographies to thereby prepare proteinaceous components that inhibit cell death.